Abstract

We evaluated the modulation by Na+,K+-ATPase
inhibitors of morphine-induced antinociception in the tail-flick test and
[3H]naloxone binding to forebrain membranes. The antinociception
induced by morphine (1-32 mg/kg, s.c.) in mice was dose-dependently
antagonized by ouabain (1-10 ng/mouse, i.c.v.), which produced a significant
shift to the right of the morphine dose-response curve. The i.c.v.
administration of three Na+,K+-ATPase inhibitors
(ouabain at 0.1-100, digoxin at 1-1,000, and digitoxin at 10-10,000 ng/mouse)
dose-dependently antagonized the antinociceptive effect of morphine (4 mg/kg,
s.c.) in mice, with the following order of potency: ouabain > digoxin >
digitoxin. This effect cannot be explained by any interaction at opioid
receptors, since none of these Na+,K+-ATPase inhibitors
displaced [3H]naloxone from its binding sites, whereas naloxone did
so in a concentration-dependent manner. The antinociception induced by
morphine (5 mg/kg, s.c.) in rats was antagonized by the i.c.v. administration
of ouabain at 10 ng/rat, whereas it was not significantly modified by
intrathecally administered ouabain (10 and 100 ng/rat). These results suggest
that the activation of Na+,K+-ATPase plays a role in the
supraspinal, but not spinal, antinociceptive effect of morphine.

Several authors, including us, have reported an activation of neuronal
Na+,K+-ATPase activity by morphine and endomorphin-1 in
vitro (Hajek et al., 1985;
Nishikawa et al., 1990;
Masocha et al., 2002;
Horvath et al., 2003) and by
morphine in vivo (Desaiah and Ho,
1977; Sharma et al.,
1998). Furthermore, it has been demonstrated that
electroacupuncture produces antinociception and a stimulation of
Na+,K+-ATPase activity that is blocked by i.p. injection
of naloxone prior to electroacupuncture
(Lee and Sun, 1984). These
results suggest a link between activation of
Na+,K+-ATPase and opioid-induced antinociception.
Na+,K+-ATPase represents the pharmacological receptor of
digitalis and strophanthus glycosides, which specifically block the activity
of this enzyme (Wallick et al.,
1979; Lingrel et al.,
1997,
1998). Therefore, if
opioid-receptor agonists stimulate Na+,K+-ATPase
activity and this action plays a role in their antinociceptive effects,
Na+,K+-ATPase inhibitors (e.g., ouabain, digoxin, and
digitoxin) might be able to antagonize such antinociceptive effects. This
hypothesis has been little tested, and discrepant results have been reported.
It has been shown that i.t. administration of ouabain enhanced
(Zeng et al., 1999) or did not
modify the antinociception induced by i.t. morphine
(Horvath et al., 2003) and
produced a small antagonism of the effect of low (but not high) doses of i.t.
endomorphin-1 (Horvath et al.,
2003). The antinociception induced by the i.t. administration of
μ-opioid agonists is mainly due to the activation of
naloxonazine-insensitive (μ2) receptors
(Paul et al., 1989;
Pasternak and Letchworth,
1999), whereas the activation of
Na+,K+-ATPase activity by morphine is
naloxonazine-sensitive (μ1-mediated)
(Masocha et al., 2002). This
prompted us to evaluate the effect of i.c.v. administration of several
Na+,K+-ATPase inhibitors (ouabain, digoxin, and
digitoxin) on the antinociception induced by the s.c. administration of
morphine, which produced a naloxonazine-sensitive antinociception
(Pick et al., 1991). Since we
found a clear antagonism of the antinociceptive effect of morphine in mice by
i.c.v.-administered digitalis glycosides, whereas all of the above-mentioned
studies that evaluate the interaction of ouabain-opioid agonists were
performed in rats with i.t. administration, we compared the effect of i.c.v.
and i.t. ouabain on the antinociception induced by s.c. morphine in rats.
Moreover, to discard the possibility that the effect of digitalis glycosides
on morphine-induced antinociception was due to a direct interaction with the
opioid receptors, we evaluated the effect of these drugs on
[3H]naloxone binding, a marker of μ-, δ-, and
κ-opioid receptors (Satoh and
Minami, 1995), to mice forebrain membranes.

Materials and Methods

Animals. The experiments were performed in CD-1 Swiss mice (Charles
River Laboratories España S.A., Barcelona, Spain) weighing 25 to 30 g
and Wistar rats (animal farm of University of Szeged, Hungary) weighing 250 to
350 g. The animals were housed in temperature-controlled (22 ± 1°C)
rooms with a 12-h light/dark cycle (lights on from 8:00 AM to 8:00 PM) and
free access to food and water. All experiments were performed during the same
period of the day (8:00 AM to 4:00 PM) to exclude diurnal variations in
pharmacological effects. The animals were randomly assigned to treatment
groups (n = 6 -15/group), and the observer was blind to the treatment
administered.

The animals were handled in compliance with European Communities Council
Directive 86/609 for the care of laboratory animals and ethical guidelines for
research in experimental pain with conscious animals
(Zimmermann, 1983). All
procedures were approved by the animal care committees.

Preparation of Mice Forebrain P2 Membranes. Mice
forebrain crude synaptosomal pellets were isolated as previously described in
detail (Gonzalez et al., 2001).
Briefly, mice forebrains were immersed in tubes containing ice-cold isolation
medium I (320 mM sucrose, 3 mM EDTA tetrasodium salt, and 10 mM HEPES, pH 7.4)
and were homogenized with a Polytron homogenizer (model PT10-35; Kinematica
AG, Basel, Switzerland). The homogenates were centrifuged (Avanti 30; Beckman
Coulter España, S. A., Madrid, Spain) at 1,000g for 10 min at
4°C; the resulting pellets were discarded, and the supernatants were
centrifuged again under the same conditions. The final supernatant was then
centrifuged at 17,000g for 20 min to obtain the crude synaptosomal
pellet (P2 pellet). Then the pellet was dissolved in the
appropriate incubation medium for binding experiments (as described under
Binding Experiments), and the protein concentration was determined by
a modified version of the Lowry et al.
(1951) method.

Binding Experiments. The P2 pellet, obtained as described
above, was dissolved in 50 mM Tris, pH 7.4. Naloxone was dissolved in 50 mM
Tris, pH 7.4, whereas ouabain, digoxin, and digitoxin were dissolved in
absolute ethanol to make up a 1-mM solution from which further dilutions were
made with buffer (50 mM Tris, pH 7.4). For measuring total binding, we
incubated in triplicate 20 μl of unlabeled drug (0.1 nM-10 μM) or its
solvent, 460 μl of P2 membrane fraction (0.6 mg/ml), and 20
μl of[3H]naloxone (2 nM) at 30°C during 30 min as previously
described (Freissmuth et al.,
1993). For measuring nonspecific binding, 10 μM of unlabeled
naloxone was added to the medium. At the end of the incubation period, the
reaction was stopped by adding 5 ml of 50 mM Tris, pH 7.4 at 4°C. Each
membrane solution was immediately filtered under vacuum through Whatman GF/B
glass fiber filters (SEMAT Technical Ltd., St. Albans, Hertfordshire, UK;
previously humidified with Tris) with a Brandel cell harvester (model M-12T;
SEMAT Technical Ltd.) and washed twice with 5 ml of 50 mM Tris HCl, pH 7.4, at
4°C. The filters were transferred to scintillation counting vials to which
4 ml of liquid scintillation cocktail (Optiphase Hisafe 2; PerkinElmer Wallac,
Loughborough, Leicestershire, UK) was added and left to equilibrate in the
dark for 12 h. The radioactivity retained on the filter was measured using a
liquid scintillation spectrometer (Beckman Coulter, Inc.) with an efficiency
of 52%. Specific binding was calculated by subtracting nonspecific binding
from total binding.

Drug Treatments and Assessment of Antinociception in Mice. Morphine
was dissolved in ultrapure water (purer than type I in the National Committee
for Clinical Laboratory Standards/College of American Pathologists water
quality standards) and injected s.c. in a volume of 5 ml/kg. The
Na+,K+-ATPase inhibitors ouabain, digoxin, and digitoxin
were dissolved in 1% Tween 80 in ultrapure water and injected i.c.v. in a
volume of 5 μl/mouse. The control animals received the same volume of
vehicles. The s.c. injections were done in the interscapular region. The
i.c.v. injections were done in the right lateral cerebral ventricle of mice
according to the method described previously
(Ocaña et al., 1993).
Briefly, the injection site was identified according to the method by Haley
and McCormick (1957), and the
drug solution was injected with a 10-μl Hamilton syringe with a sleeve
around the needle to prevent the latter from penetrating more than 3 mm into
the skull. After the experiments were done, the position of the injection was
evaluated in each brain, and the results from animals in which the tip of the
needle did not reach the lateral ventricle were discarded.

The antinociceptive effect of the treatments was evaluated using a
tail-flick test as previously described
(Ocaña et al., 1993).
Briefly, the animals were restrained in a Plexiglas tube and placed on the
tail-flick apparatus (LI 7100; Letica SA, Barcelona, Spain). A noxious beam of
light was focused on the tail about 4 cm from the tip, and the latency to
tail-flick was recorded automatically to the nearest 0.1 s. The intensity of
the radiant heat source was adjusted to yield baseline latencies between 3 and
5 s; this intensity was never changed, and any animal whose baseline latency
was outside the pre-established limits was excluded from the experiments. Two
baseline tail-flick latencies were recorded within 20 min before all
injections. At time 0, the animals received an i.c.v. injection of ouabain,
digoxin, digitoxin, or their solvent and immediately thereafter an s.c.
injection of morphine or its solvent. The end of the last injection was
considered as time 0; from this time, tail-flick latencies were measured again
at 10, 20, 30, 45, 60, 90, and 120 min after treatment. The cutoff time was 10
s.

The area under the curve (AUC) of tail-flick latency against time was
calculated for each animal using the GraphPad Prism version 3.00 for Windows
(GraphPad Software Inc., San Diego, CA). The degree of antinociception was
determined according to the formula: % antinociception = [(AUCd -
AUCv) / (AUCmax - AUCv)] × 100, where
the AUCd and AUCv are the areas under the curve for
drug- and vehicle-treated mice, respectively, and AUCmax is the
area under the curve of maximum possible antinociception (10 s in each
determination).

Drug Treatments and Assessment of Antinociception in Rats. Morphine
was dissolved in saline and injected s.c. in a volume of 2 ml/kg. Ouabain was
dissolved in 1% Tween 80 in water and injected i.t. or i.c.v. in a volume of 5
μl/rat. Control animals received the same volume of the vehicles. For the
intrathecal drug administration, the rats were surgically prepared under
ketamine-xylazine anesthesia (72 and 8 mg/kg intraperitoneally, respectively).
An intrathecal catheter (PE-10 tubing) was inserted through a small opening in
the cisterna magna and passed 8.5 cm caudally into the intrathecal space as
previously described (Yaksh and Rudy,
1976). For i.c.v. drug administration, the rats were anesthetized
as described above and placed in a stereotaxic apparatus. A stainless steel
23-gauge cannula was placed into the right lateral ventricle and fixed to the
skull with dental cement. Coordinates were 0.5 mm caudal and 1.5 mm lateral to
the bregma, with the cannula extending 3.5 mm ventral to the skull surface.
Injections were performed through a 29-gauge internal cannula, which extended
1.0 mm beyond the guide cannula tip. The internal cannula was attached to a
Hamilton 10-μl syringe with a polyethylene tubing (PE-20), allowing the
animals to move freely during the injection period. After surgery (both i.t.
and i.c.v.), the rats were housed individually, had free access to food and
water, and were allowed to recover for at least 4 days before use. Rats
exhibiting postoperative neurologic deficits were not used. Each animal was
studied twice in an experimental series, with an interval of 7 to 8 days
between studies. After experimental use, each rat was killed with an overdose
of pentobarbital, and 1% methylene blue was injected to confirm the position
of the catheter (i.t.) or cannula (i.c.v.) and probable spread of the
injection. If the position was not right, the results obtained in the animal
were discarded.

The antinociceptive effect of the treatments was evaluated using a
tail-flick test as previously described
(Horvath et al., 1990). The
reaction time was determined by immersing the lower 5-cm portion of the tail
in hot water (51.5°C) until a tail-withdrawal response was observed. The
basal latency was 8.9 ± 0.32 s, and the cutoff time was 20 s. The
tail-flick latencies were recorded immediately before and at 10, 30, 60, 90,
and 120 min after the drug injections of morphine alone or associated to
ouabain or its' solvent. The AUC of tail-flick latency against time and the
degree of antinociception in each animal were calculated as described in the
previous section but using 20 s in each determination to calculate the
AUCmax. In the experiments where the effect of ouabain alone was
tested, the drug was injected cumulatively (1, 10, 100, and 1,000 ng).

Data Analysis. The values of IC50 (concentration of
unlabeled drug that inhibited 50% of specific [3H]naloxone
binding), ED50 (dose of morphine that produced half of the maximal
antinociception), and Emax (maximum antinociception
produced) were calculated from the concentration-response curves or
dose-response curves using nonlinear regression analysis with GraphPad Prism
version 3.00. Statistical analysis was performed using one-way analysis of
variance (ANOVA), followed by Newman-Keuls multiple comparison test or two-way
ANOVA, followed by Bonferroni post test, and the differences were considered
significant when p < 0.05. The results in the text and figures are
expressed as the means ± S.E.M.

Results

Effect of i.c.v. Ouabain on the Antinociception Induced by Morphine in
Mice. The analysis of AUC values showed that the administration of
morphine (1-32 mg/kg, s.c.) together with the vehicle of ouabain (i.c.v.)
produced a dose-dependent increase in the percentage of antinociception in the
treated mice (Fig. 1). Using
nonlinear regression analysis, we fitted the data to a sigmoid curve, which
made it possible to calculate Emax as 78.40 ± 2.94%
of antinociception and ED50 as 2.16 ± 0.20 mg/kg. The
treatment of mice with ouabain (1 and 10 ng/mouse, i.c.v.) produced a
dose-dependent antagonism of the ability of morphine to induce antinociception
and displaced the dose-response curve of morphine to the right
(Fig. 1). The maximum efficacy
of morphine was dose-dependently reduced by ouabain (Emax
= 67.49 ± 2.19 and 63.45 ± 1.58% for mice treated with 1 and 10
ng/mouse, respectively). The ED50 of morphine was dose-dependently
increased in the animals treated with ouabain (ED50 = 5.02 ±
0.34 and 6.43 ± 0.34 mg/kg for mice treated with 1 and 10 ng/mouse,
respectively).

Antagonism by treatment with ouabain (1 and 10 ng/mouse, i.c.v.) of the
antinociception induced by morphine (1-32 mg/kg, s.c.) in a tail-flick test in
mice. The percentage of antinociception was calculated from the area under the
curve of tail-flick latency along time (as described under Materials and
Methods). Each point represents the mean ± S.E.M of the values
obtained from 7 to 14 animals. Statistically significant differences in
comparison with morphine + vehicle: *, p < 0.05 and
**, p < 0.01 (two-way ANOVA followed by Bonferroni
test).

The s.c. administration of morphine (4 mg/kg) together with the i.c.v.
injection of the vehicle of ouabain induced an increase in tail-flick latency
in a time-dependent manner (Fig.
2, left side). The maximum effect was observed at 30 min and
posteriorly decayed progressively. Ouabain (0.1-10 ng/mouse, i.c.v.)
dose-dependently antagonized this effect of morphine; the antagonism was
particularly evident from 30 to 120 min after morphine administration
(Fig. 2, left side). The
administration of ouabain (0.1-100 ng/mouse, i.c.v.) together with the solvent
of morphine (s.c.) did not significantly modify tail-flick latency at any of
the times and doses tested (data not shown).

Antagonism by i.c.v. treatment with ouabain of the antinociception induced
by morphine (4 mg/kg, s.c.) in a tail-flick test in mice. Left side, time
course of the tail-flick latency times for various combinations of morphine (4
mg/kg, s.c.) or its vehicle and ouabain (0.1, 1, and 10 ng/mouse, i.c.v.) or
its vehicle. Each point represents the mean ± S.E.M of the values
obtained from 10 to 14 animals. Statistically significant differences in
comparison with morphine + vehicle: *, p < 0.05 and
**, p < 0.01 (two-way ANOVA followed by Bonferroni
test). Right side, antagonism by treatment with ouabain (0.1-100 ng/mouse,
i.c.v.) of the antinociception induced by morphine (4 mg/kg, s.c.). The solid
column represents the effect of morphine + ouabain vehicle. The percentage of
antinociception was calculated from the area under the curve of tail-flick
latency along time (as described under Materials and Methods). Each
column represents the mean ± S.E.M of the values obtained from 10 to 14
animals. Statistically significant differences in comparison with morphine +
vehicle: *, p < 0.05 and **, p <
0.01 (one-way ANOVA followed by Newman-Keuls test).

When the AUC of antinociception along time was analyzed, ouabain (0.1-10
ng/mouse, i.c.v.) antagonized the antinociceptive effect of morphine (4 mg/kg,
s.c.) in a dose-dependent manner (Fig.
2, right side). The maximum antagonism by ouabain was produced at
10 ng/mouse, which reduced the percent antinociception from 69 ± 7.4%
(morphine + vehicle) to 20 ± 8.9%. A higher dose of ouabain (100
ng/mouse, i.c.v.) produced a slightly lower antagonism of morphine
antinociception (Fig. 2, right
side).

Effects of Several Na+,K+-ATPase
Inhibitors on the Antinociception Induced by Morphine in Mice. The s.c.
administration of morphine (4 mg/kg) together with the vehicle of the
Na+,K+-ATPase inhibitors (i.c.v.) produced a percentage
of antinociception of 74.77 ± 6.75%
(Fig. 3). The antinociception
induced by morphine (4 mg/kg, s.c.) was dose-dependently antagonized by the
i.c.v administration of the three Na+,K+-ATPase
inhibitors (ouabain at 0.1 to 100, digoxin at 1 to 1,000, and digitoxin at 10
to 10,000 ng/mouse), with the following order of potency: ouabain > digoxin
> digitoxin. As seen in Fig.
3, the Na+,K+-ATPase inhibitors did not
antagonize completely the antinociception produced by morphine (4 mg/kg,
s.c.). The least percentage of antinociception produced by morphine (4 mg/kg,
s.c.) associated with the highest dose of the
Na+,K+-ATPase inhibitor used was 28.61 ± 7.08%
for ouabain (100 ng/mouse, i.c.v.), 37.08 ± 5.7% for digoxin (1,000
ng/mouse, i.c.v.), and 28.97 ± 9.46% for digitoxin (10,000 ng/mouse,
i.c.v.). Nonstatistically significant differences were found between these
values of antinociception. Higher doses of Na+,K+-ATPase
inhibitors were not tested, as they caused hyperexcitability and convulsions
in some animals.

Antagonism by i.c.v. treatment with different doses of ouabain, digoxin,
and digitoxin of the antinociception induced by morphine (4 mg/kg, s.c.) in a
tail-flick test in mice. The percentage of antinociception was calculated from
the area under the curve of tail-flick latency along time (as described under
Materials and Methods). The shaded area represents the mean ±
S.E.M of the antinociception produced in the animals treated with morphine +
vehicle. Each point represents the mean of the values obtained from 6 to 12
animals. Statistically significant differences in comparison with morphine +
vehicle: *, p < 0.05 and **, p <
0.01 (one-way ANOVA followed by Newman-Keuls test).

Effects of Na+,K+-ATPase Inhibitors
and Naloxone on [3H]Naloxone Binding to Mice Forebrain
Membranes.Fig. 4 shows the
competition for [3H]naloxone binding sites by naloxone and the
Na+,K+-ATPase inhibitors (ouabain, digoxin, and
digitoxin). Naloxone (0.1 nM-10 μM) concentration-dependently displaced
[3H]naloxone from its binding sites to forebrain membranes, with an
IC50 of 3.58 ± 0.18 nM. In contrast, ouabain, digoxin, and
digitoxin, even at concentrations of 10 μM, did not significantly modify
[3H]naloxone-specific binding.

Displacement by naloxone but not by Na+,K+-ATPase
inhibitors (ouabain, digoxin, and digitoxin) of [3H]naloxone (2 nM)
binding to mice forebrain membranes. Each point represents the mean ±
S.E.M. of the values from three independent experiments.

Effect of i.c.v. Administration of Ouabain on the Antinociception
Induced by Morphine in Rats. The s.c. administration of morphine (5 mg/kg)
together with the i.c.v. injection of the vehicle of ouabain induced an
increase in tail-flick latency in a time-dependent manner. The maximum effect
was observed at 30 min and posteriorly decayed slowly
(Fig. 5, left side). Ouabain
(10 ng/rat, i.c.v.) antagonized this effect of morphine; the antagonism was
evident from 30 to 120 min after morphine administration, although only
reaching statistical significance at 90 and 120 min. On the other hand, a
higher dose of ouabain (100 ng/rat, i.c.v.) did not significantly antagonize
morphine-induced antinociception at any time
(Fig. 5, left side). The
administration of ouabain (10 and 100 ng/rat, i.c.v.) together with the
solvent for morphine (s.c.) did not modify the tail-flick latency at any of
the doses or times tested (data not shown). When the area under the curve of
antinociception along time was analyzed, ouabain (10 ng/rat, i.c.v.)
significantly antagonized the antinociceptive effect of morphine (5 mg/kg,
s.c.), whereas the highest dose of ouabain used (100 ng/rat, i.c.v.) did not
(Fig. 5, right side).

Antagonism by i.c.v. treatment with ouabain of antinociception induced by
morphine (5 mg/kg, s.c.) in a tail-flick test in rats. Left side, time course
of the tail-flick latency times for various combinations of morphine (5 mg/kg,
s.c.) or its vehicle and ouabain (10 and 100 ng/rat, i.c.v.) or its vehicle.
Each point represents the mean ± S.E.M of the values obtained from 6 to
10 animals. Statistically significant differences in comparison with morphine
+ vehicle: **, p < 0.01 (two-way ANOVA followed by
Bonferroni test). Right side, antagonism by treatment with ouabain (10 and 100
ng/rat, i.c.v.) of the antinociception induced by morphine (5 mg/kg, s.c.).
The solid column represents the effect of morphine + ouabain vehicle. The
percentage of antinociception was calculated from the area under the curve of
tail-flick latency along time (as described under Materials and
Methods). Each column represents the mean ± S.E.M of the values
obtained from 6 to 10 animals. Statistically significant differences in
comparison with morphine + vehicle: *, p < 0.05
(one-way ANOVA followed by Newman-Keuls test).

Effect of i.t. Administration of Ouabain on the Antinociception Induced
by Morphine in Rats. The administration of morphine (5 mg/kg, s.c.)
together with the vehicle of ouabain induced an increase in tail-flick latency
that reached a maximum at 30 min and decreased at 120 min
(Fig. 6, left side). Ouabain
(10 ng/rat, i.t.) significantly antagonized this effect of morphine only at 60
min after drug administration. On the other hand, a higher dose of ouabain
(100 ng/rat, i.t.) had a significant synergistic effect with morphine (5
mg/kg, s.c.) only at 10 min after drug administration
(Fig. 6, left side). When the
area under the curve of antinociception along time was analyzed, none of the
doses of ouabain (10 and 100 ng/rat, i.t.) significantly modified the
antinociceptive effect of morphine (5 mg/kg, s.c.)
(Fig. 6, right side). The
administration of ouabain (10 and 100 ng/rat, i.t.) together with the solvent
of morphine (s.c.) did not modify tail-flick latency at any of the doses
tested (data not shown).

Effect of i.t. treatment with ouabain on the antinociception induced by
morphine (5 mg/kg, s.c.) in a tail-flick test in rats. Left side, time course
of the tail-flick latency times for various combinations of morphine (5 mg/kg,
s.c.) or its vehicle and ouabain (10 and 100 ng/rat, i.t.) or its vehicle.
Each point represents the mean ± S.E.M of the values obtained from 7 to
10 animals. Statistically significant differences in comparison with morphine
+ vehicle: *, p < 0.05 and **, p
< 0.01 (two-way ANOVA followed by Bonferroni test). Right side, effect of
treatment with ouabain (10 and 100 ng/rat, i.t.) on the antinociception
induced by morphine (5 mg/kg, s.c.). The solid column represents the effect of
morphine + ouabain vehicle. The percentage of antinociception was calculated
from the area under the curve of tail-flick latency along time (as described
under Materials and Methods). Each column represents the mean
± S.E.M of the values obtained from 7 to 10 animals. No statistically
significant differences were observed in comparison with morphine + vehicle
(one-way ANOVA).

Discussion

The present study found two main results: 1) that ouabain, digoxin, and
digitoxin did not have affinity for opioid receptors but when i.c.v.
administered antagonized the antinociceptive effect of s.c. morphine in mice;
and 2) that ouabain administered i.c.v., but not i.t., antagonized the
antinociception induced by s.c. morphine in rats.

The i.c.v. administration of several digitalis glycosides dose-dependently
antagonized the antinociceptive effect of morphine in mice. Under the same
experimental conditions, these drugs, when i.c.v. injected (ouabain at 10 and
100, digoxin at 100 and 1000, and digitoxin at 1000 and 10000 ng/mouse), did
not modify the antinociceptive effect of the κ-opioid receptor agonist
trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide
methanesulfonate (8 mg/kg, s.c.) (manuscript in preparation). This indicates
that the antagonism by digitalis glycosides of morphine-induced
antinociception is specific and not an indiscriminate antagonism of the
antinociception induced by any drug.

The antagonism of the antinociceptive effect of morphine by digitalis
glycosides cannot be explained by a direct interaction of these drugs with the
opioid receptors, since none of these drugs significantly displaced
[3H]naloxone, a marker of μ-, δ-, and κ-opioid
receptors (Satoh and Minami,
1995), from its binding sites to mice forebrain membranes. On the
other hand, morphine increases Na+,K+-ATPase activity
(Nishikawa et al., 1990;
Sharma et al., 1998;
Masocha et al., 2002), and
digitalis glycosides specifically block the activity of this enzyme
(Wallick et al., 1979; Lingrel
et al., 1997,
1998); therefore, the ability
of digitalis glycosides to antagonize morphine-induced antinociception can be
due to their ability to block the Na+,K+-ATPase
activity. In support of this hypothesis, it is interesting to note that the
order of potency of the digitalis glycosides in antagonizing morphine-induced
antinociception (ouabain > digoxin > digitoxin) is the same as their
order of potency in inhibiting [3H]ouabain binding in brain
membranes (Acuña-Castroviejo et al.,
1992). The fact that about 30% of the antinociception induced by
morphine in mice was insensitive to digitalis glycosides might be due to the
fact that beside the stimulation of Na+,K+-ATPase
activity, other mechanisms, like the opening of KATP channels
(Ocaña et al., 1993,
1995;
Raffa and Martinez, 1995), the
closing of voltage-dependent Ca2+ channels
(Del Pozo et al., 1990;
Dierssen et al., 1990), or the
inhibition of adenylate cyclase (Suh et al.,
1995,
1996), are involved in the
antinociceptive effect of morphine. Another possible reason to explain the
partial antagonism by digitalis glycosides of morphine-induced antinociception
might be related to the different routes of administration of both drugs. When
morphine is administered peripherally (s.c. or i.p.), it is distributed in
supraspinal and spinal sites (Miyamoto et
al., 1991), and the activation of opioid receptors located in both
sites is essential for the production of its antinociceptive effect (Yeung and
Rudy,
1980a,b;
Roerig and Fujimoto, 1989).
When digitalis glycosides are i.c.v. administered, they are expected to be
mainly distributed in the supraspinal space. Then, the portion of
morphine-induced antinociception not blocked by i.c.v.
Na+,K+-ATPase inhibitors could have been, at least
partially, spinally mediated. In support of this notion, we found that i.t.
administration of ouabain did not antagonize the antinociception induced by
i.t. morphine in rats (Horvath et al.,
2003) nor that produced by s.c. morphine in rats (present
study).

In agreement with the data obtained in mice, we observed that the i.c.v.
administration of ouabain (10 ng/rat) reduced the antinociception induced by
morphine in rats. However, the antagonism of morphine-induced antinociception
produced by i.c.v. administration of ouabain was less in rats than in mice.
This might be attributed to the species-specific differences of the
Na+,K+-ATPase α-subunit
(Feschenko et al., 1997) and
variations between species in residues that convey ouabain sensitivity within
the first and second transmembrane helices hairpin of the
Na+,K+-ATPase
(Lingrel et al., 1997). It
could also be speculated that, in the lower degree of antagonism by i.c.v.
ouabain in rats compared with mice, a greater relative contribution of spinal
versus supraspinal sites of action for s.c. morphine-induced antinociception
in rats than mice might have played a role. On the other hand, it is
interesting to note that a higher dose of ouabain (100 ng/rat) produces less
antagonism of morphine effect than 10 ng/rat. A similar fact was observed in
mice (see Fig. 2, right side),
which suggests that a relatively high dose of ouabain may produce effects
additional to those of low doses that can counteract the antagonism induced by
the low doses of ouabain. In fact, the only study that has evaluated
previously the effect of i.c.v. ouabain on morphine-induced antinociception
found that 100 ng of ouabain did not antagonize the effect of morphine
(Calcutt et al., 1971).

On the other hand, 10 ng/rat ouabain i.t. reduced the increase of
tail-flick latency induced by morphine only at 60 min after drug
administration, whereas a higher dose of ouabain (100 ng/rat, i.t.) had a
synergistic effect on the morphine-induced increase in tail-flick latency only
at 10 min after drug administration. However, both doses of ouabain (10 and
100 ng/rat, i.t.) had no significant effect on the antinociception induced by
morphine in rats when a global value of antinociception (AUC of tail-flick
latency along time) was used. These results demonstrate a difference between
the effects of i.c.v.- and i.t.-administered ouabain on the morphine-induced
antinociception. Previous studies have reported that i.t.-administered ouabain
enhanced (Zeng et al., 1999)
or did not modify the antinociception induced by i.t. morphine
(Horvath et al., 2003).
Therefore, it seems that ouabain antagonized the supraspinal antinociception
induced by morphine, whereas it did not significantly antagonize its spinal
antinociception. The supraspinal antinociceptive effects of morphine are
mediated mainly through μ1 opioid receptors, whereas the spinal
antinociceptive effects are mediated through μ2 opioid receptors
(Paul et al., 1989;
Pasternak and Letchworth,
1999). We have demonstrated that μ1-opioid receptors
have an important role in the enhancement of synaptosomal
Na+,K+-ATPase activity induced by morphine
(Masocha et al., 2002). Thus,
one would expect an antagonism of the antinociceptive effect of morphine by
ouabain administered i.c.v. but not i.t.

In conclusion, our results suggest that activation of neuronal
Na+,K+-ATPase plays a role in the supraspinal, but not
spinal, antinociceptive effect of morphine.

Acknowledgments

W. M. was supported by grants from the Agencia Española de
Cooperación Internacional and the University of Granada.

Footnotes

This work was supported by a Hungarian-Spanish Scientific Research grant
(17/2001), Spanish Scientific Research Council grants from Comisión
Inter-ministerial de Ciencia y Tecnológica (SAF 97-0173) and Junta de
Andalucía (CTS-109), and Hungarian Scientific Research grants (OTKA
T-34741 and ETT 042/2001).